The promise of stem cell biology is predicated on the idea that these cells will be utilized in regenerative medicine and to model development and disease of tissues in vitro. While fantastic progress has been made in the generation of patient-specific stem cells through the use of reprogramming of somatic cells to a pluripotent state, little is known about the basic biology of these cells and whether they will be able to live up to their promise in regenerative medicine, development and disease. We have found that in vitro differentiation from hESCs and hiPSCs does not accurately reflect in vivo differentiation in human tissue. We have identified a molecular basis for this discrepancy and preliminary data suggest that in fact differentiation from human pluripotent stem cells could reflect human development but from a very early embryonic stage. We identified LIN28/let-7 as able to control human differentiation during early fetal development, in congruence with what has been shown for this circuit in C. Elegans. We propose to manipulate this important gene expression circuit to determine whether proper control of this circuit is vital to allow for maturation of human cells. This project will also develop tools that will allow for manipulation of this circuit and a simplified method for driving cellular maturation in vitro. Finally, we will use genome-wide approaches to understand how this circuit is normally regulated and whether it is representative of a cohort of co-regulated genes important for human development. This project will significantly contribute to our limited knowledge of human development, and the control of cell fate with human pluripotent stem cells. There will be no human subjects or animal experimentation with the proposed work.